The project focuses on the elucidation of molecular mechanisms by which the immunologically important NF-kappaB transcription factors can be activated. In addition to leading to a better basic understanding of signaling processes, identification of the molecular components of signaling pathways will also reveal potential targets for therapeutic intervention to block activation of NF-kappaB in various diseases. Inflammatory diseases, for example, are often driven by the undesirable activation of NF-kappaB. In addition, expression of the human immunodeficiency virus (HIV) and other clinically relevant viruses depends in large part on activation of NF-kappaB, making these transcription factors and their regulators potential targets for controlling the spread of HIV and other viruses. NF-kappaB transcription factors are normally retained in the cytoplasm by association with the inhibitory IkappaB proteins. Signals directly or indirectly related to pathogens or stress lead to phosphorylation and then proteolytic degradation of the inhibitor, thereby releasing the NF-kappaB factors to translocate to the nucleus and carry out their functions. Additional regulation is imposed via direct or indirect effects of signals on the transactivation activity of these factors once they have entered the nucleus. The present project is concerned with the delineation of several signaling paths that lead to phosphorylation and subsequent ubiquitin-dependent proteasomal degradation of the IkappaB inhibitors as well as those paths that stimulate transactivation. We have previously cloned CIKS, as a protein associated with NEMO, the regulatory subunit of the core IkappaB kinase (IKK) complex. In addition to NEMO, the IKK core complex also contains the IkappaB kinases alpha and beta. Transfected CIKS not only activates NF-kappaB, but also the Jun Kinase. We now describe another adaptor-like protein, TANK, which can also associate with IKKs via NEMO. TANK in turn provides a link between the IKK complex and the kinases IKKepsilon and TBK1. The existence of such large complexes surrounding the IKKs may allow the IKKepsilon and TBK1 kinases to influence IKK-mediated phosphorylation of IkappaB inhibitory proteins and/or the transactivation potential of the associated NF-kappaB complexes. Proteins such as CIKS and TANK are likely to be critical for funneling specific signals to the IKK kinases. In addition to the dissection of pathways that lead to the activation of NF-kappaB via the classical route, namely the induced degradation of small IkappaBs, we have also begun to dissect an alternative pathway of activation of NF-kappaB. This second pathway involves processing of the p100 precursor form of NF kappaB2 to the p52 form, which in particular leads to activation of p52/RelB heterodimers. We have identified a physiologic inducer of this second activation pathway, the TNF family member BAFF which targets B cells. We have furthermore determined that this pathway depends on the IKKalpha subunit of the IKK complex and on the NF-kappaB inducing kinase, but that it does not require the IKK subunits NEMO or IKKbeta. Therefore the second pathway is independent of the classical IKK complex.